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Active and regulatory sites of cytosolic 5′-nucleotidase

Pesi, Rossana and Allegrini, Simone and Careddu, Maria Giovanna and Filoni, Daniela Nicole and Camici, Marcella and Tozzi, Maria Grazia (2010) Active and regulatory sites of cytosolic 5′-nucleotidase. FEBS Journal, Vol. 277 (23), p. 4863-4872. eISSN 1742-4658. Article.

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DOI: 10.1111/j.1742-4658.2010.07891.x


Cytosolic 5′-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap4A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap4A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation.

Item Type:Article
ID Code:5683
Uncontrolled Keywords:cN-II active site, cN-II regulatory sites, cN-II structure, cytosolic 5′-nucleotidase II
Subjects:Area 05 - Scienze biologiche > BIO/10 Biochimica
Divisions:001 Università di Sassari > 01 Dipartimenti > Scienze del farmaco
Publisher:Blackwell / Wiley
Copyright Holders:© 2010 The Authors , Journal compilation © 2010 FEBS
Deposited On:01 Mar 2011 09:49

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